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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Fluorimetric HDAC Activity Assay Kit *Green Fluorescence*/13601/200 Tests
产品编号:13601
市  场 价:¥56560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Fluorimetric HDAC Activity Assay Kit *Green Fluorescence*/13601/200 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)498/520
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
Histonedeacetylase(HDAC)
RelatedMicroplateReaders
BiochemicalAssays
Histonedeacetylases(HDAC)areaclassofenzymesthatremoveacetylgroupsfroma?-N-acetyllysineaminoacidonahistone.Deacetylationrestoresthepositiveelectricchargeofthelysineaminoacids,whichincreasesthehistone"saffinityforthenegativelychargedphosphatebackboneofDNA.Thisgenerallydown-regulatesDNAtranscriptionbyblockingtheaccessoftranscriptionfactors.HDACsareinvolvedinthepathwaybywhichtheretinoblastomaprotein(pRb)suppressescellproliferation.ThepRbproteinispartofacomplexwhichattractsHDACstothechromatinsothatitwilldeacetylatehistones.HDACinhibitorsarebeingstudiedasatreatmentforcancer.TheAmplite™HDACAssayKitprovidesaquick,convenient,andsensitivemethodforthedetectionofHDACactivity.OurHDACGreen™substrateisanon-peptidecompoundthatismuchmoreresistantthanothercommercialpeptide-basedHDACsubstrates.OurkitcanbeusedformeasuringHDACactivityincelllysates,invitroinhibitorscreeningwithextractsorpurifiedenzymes.ThelongwavelengthemissionandhigherextinctioncoefficientoftheHDACGreen™substrateprovidelessinterferencefromcompoundsandcellcomponents.HDACactivityisdeterminedbymonitoringthegreenfluorescenceenhancementwithexcitationat490nmandemissionat520nm.
SpectrumAdvancedSpectrumViewer

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Movemouseovergridtodisplaywavelength&intensityvalues.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepareworkingsolution:

1.1   PrepareHDAC-containingtestsamples:Dilute5–10mg/mLofHeLanuclearextractorcelllysatesat1:40inAssayBuffer(ComponentB).

Note:40µLofthedilutedsampleisenoughforonewellofa96-wellplate.Diluteextractimmediatelybeforeuse.Storethesolutiononice.

 

1.2   PreparedilutionsofHDACinhibitor(TrichostainA)solution:Dilute3mMTrichostatinAsolution(ComponentC)at1:100inassaybuffer(ComponentB)togeta30μMTrichostatinAsolution.Add10μLofthe30μMTrichostatinAsolutionintoeachinhibitorcontrolwell.

 

1.3   PrepareHDACGreen™Substrateworkingsolution:Add20µLofHDACGreen™Substrate(ComponentA)and100µLoftheSignalEnhancer(ComponentD)into5mLofAssayBuffer(ComponentB).

Note1:ThedilutedHDACGreen™Substrateworkingsolutionisnotstable,5mLofthedilutedHDACGreen™Substrateworkingsolutionisenoughfor100assays.

Note2:PreparefreshHDACGreen™Substrateworkingsolutionforeachexperiment.Keepreconstitutedworkingsolutiononiceuntiluse.

 

2.RunHDACAssay:

2.1   Add40μLofdilutednuclearextract,enzymesolutionorotherHDACsamplesand10μLoftestcompoundstothecorrespondingmicroplatewells(seeTable1).

Forpositivecontrol:Add40μLofdilutedHDACenzymesolutionorHeLanuclearextract(fromStep1.1)with10μLofAssayBuffer(ComponentB).

Fornegativecontrol:Add40μLofdilutedHeLanuclearextract(fromStep1.1)with10μLof30μMTrichostatinAsolution(fromStep1.2),oruseaknownsamplecontainingnoHDACactivity.

ForBlank(noEnzyme):Add50μLofAssayBuffer(ComponentB)only.

 

2.2   Incubatetheplateatroomtemperatureor37Cfor10-20minutes.

Note:ForscreeningHDACinhibitor,preincubatethecompoundswithHeLanuclearextractorpureenzymebeforeaddingHDACGreen™Substrateworkingsolution(seeStep2.3)

 

2.3   Add50μLofHDACGreen™Substrateworkingsolution(fromStep1.3)intoeachwell.Incubatetheplateatroomtemperatureor37oCfor30-60minutes.

 

2.4   MonitorfluorescenceintensityatEx/Em=490/525nm.

 

Table1.Layoutofnuclearextractswithtestcompoundsinasolidblack96-wellmicroplate

 

Samples

HeLaExtract

(fromStep1.2)

AssayBuffer

(ComponentB)

TrichostatinA

(fromStep1.3)

TestCompounds

HDACGreen™Substrate(fromStep1.1)

Blank(noEnzyme)

0µL

50µL

0µL

0µL

50µL

PositiveControl

40µL

10µL

0µL

0µL

50µL

NegativeControl

40µL

0µL

10µL

0µL

50µL

TestCompounds

40µL

0µL

0µL

10µL

50µL

References&Citations
CitationExplorer

Marine-derivechromopeptideA,anovelclassIHDACinhibitor,suppresseshumanprostatecancercellproliferationandmigration
Authors:Jing-yaSun,Ji-dongWang,XinWang,Hong-chunLiu,Min-minZhang,Yu-ChihLiu,Chen-huaZhang,YiSu,Yan-yanShen,Yue-weiGuo
Journal:ActaPharmacologicaSinica(2017)

Butyrateupregulatesendogenoushostdefensepeptidestoenhancediseaseresistanceinpigletsviahistonedeacetylaseinhibition
Authors:HaitaoXiong,BingxiuGuo,ZhenshunGan,DeguangSong,ZeqingLu,HongboYi,YuemingWu,YizhenWang,HuahuaDu
Journal:Scientificreports(2016)

Software-programmablecontinuous-flowmulti-purposelab-on-a-chip
Authors:AhmedMAmin,RavirajThakur,SethMadren,Han-ShengChuang,MithunaThottethodi,TNVijaykumar,StevenTWereley,StephenCJacobson
Journal:Microfluidicsandnanofluidics(2013):647--659


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